A large variety of body implants are known for medical uses such as substitute vascular prostheses, skin dressings and coverings, and for other purposes. The implant material can be synthetic or body tissues from the same species or other species as the species to be implanted. When body tissues and structures are to be implanted, they may be used fresh from the donor but in many cases, it is preferred to have some means of preserving the implant tissue for later use. There are several preservation techniques currently available including cryopreservation and chemical fixation with cross-linking agents such as glutaraldehyde, polyglycidyl ether and carbodiimide. In order to prepare the implant tissue for later use it is desirable to decellularise the tissue prior to storage whilst minimising any damage to the physical structure of the tissue matrix itself. This decellularisation can be important in improving the biocompatability and reducing the immunological reaction in the tissue graft.
It is known from the prior art to use anionic detergents such as sodium dodecyl sulphate (SDS) for the extraction of cellular components. SDS extraction was first described in U.S. Pat. No. 4,323,358 as a method of preventing or delaying the calcification of glutaraldehyde-fixed Hancock heart valve bioprosthesis, the method is referred to as the “Hancock T6 treatment”. In this method, fixed tissue is contacted with SDS so as to retard calcification. However, serious limitations of the method have been reported (Bodnar E et al, Thorac. Cardiovasc. Surgeon.1985 34: 82-85; Courtman D Wet al, J Biomed Mater Res. 1994 28: 655-666; Wilson G J et al. ASAIO Trans 1990 36: M340-343). These researchers all report that SDS has a deleterious effect on heart valve extracellular matrix (ECM) and in particular on the collagen and elastin fibre components.
In order to mitigate the effects of SDS on ECM, U.S. Pat. No. 4,776,853 describes the use of an earlier pre-treatment with other non-ionic detergents, such as Triton X-100 so that SDS is only employed as the second detergent in a multistage detergent decellularisation program.
A further problem associated with decellularising tissue implant is to minimise the degradation to the ECM during the process. It is known to use protease inhibitors to prevent such degradation during incubation with a non-ionic detergent in the first stage of the multistage detergent decellularisation program and also to use them to prevent naturally occurring proteases from attacking collagens. There are a number of different proteases that reside within the tissue matrix, either in direct association with the cells themselves or bound within the ECM. One of the largest of the protease families, the matrix metalloproteases (MMPs), has a wide range of substrate specificities including collagen, laminin, fibronectin and elastin. Another important family of matrix-degrading proteases are the plasminogen activators, which generates the broad specificity protease plasmin from the abundant zymogen plasminogen. As well as proteolytic activity, plasmin has the further ability of activating members of the MMP family. However, most of the protease inhibitors are inherently toxic which is undesirable if the matrix is to be seeded with living cells and implanted into a human or animal. Moreover, some of the protease inhibitors used so far, for example PMSF, are extremely unstable in solution having a half life of less than 1 hour, and since decellularisation is a lengthy process i.e. several days, this limits the choice of inhibitors that have sufficient half lives.
A method which could simplify the decellularisation process whilst minimising damage to ECM would offer significant advantage over current practices.